Project Summary Cancer remains a major killer that impacts a large number of new patients every year. One leading reason for cancer is an inappropriate response to DNA damage. Upon DNA damage, the cell will initiate apoptosis through the mitochondria apparatus when DNA damage is beyond repair. Once this cell death pathway is triggered, cytotoxic factors are released from mitochondria to activate caspases that induce apoptosis. An impaired apoptotic potential of damaged cells leads to continued cell division without restriction that frequently develops into cancer. Indeed, mis-regulation of apoptosis is associated with many cancers. Understanding the molecular mechanisms that regulate DNA damage signals and how they are transmitted to mitochondria to initiate apoptosis is therefore important for cancer research. Biochemical approaches are utilized to reconstitute DNA damage-induced apoptosis. Degradation of Mcl-1, a key anti-death protein, is required to trigger cytochrome c release from mitochondria upon DNA damage. Mcl-1 is degraded through the ubiquitin-proteasome pathway. In the ubiquitin-proteasome system, the ubiquitin ligases determine the specificity and timing of substrates destruction. A biochemical assay was established to search for such enzyme from human cell extracts, and a novel ubiquitin ligase Mule (Mcl-1 ubiquitin ligase e3) was cloned. Mule promotes ubiquitin modification of Mcl-1 through direct interaction through a BH3 domain, and Mule is indispensable for Mcl-1 mediated apoptotic pathway. Besides Mcl-1, Mule also ubiquitinates other substrates including p53, thus adding another intriguing link to the apoptosis pathway. In this proposal, the mechanism by which Mule activation and the differential regulation of Mule target different substrates in response to DNA damage signals will be investigated. The biological function of Mule in DNA damage response will be studied in Aim 1. Our newly generated Mule knockout mouse embryonic fibroblast cells will be utilized to study the function of Mule in DNA damage induced apoptosis and cell cycle checkpoints. We will also investigate the structure and function relationship of Mule, especially the requirement of the ubiquitin ligase activity of Mule for its function in DNA damage response in this aim. We will characterize the ubiquitination and degradation of Mcl-1 and p53 by Mule in Aim 2. Ubiquitin chain formation, E3 activity (Mule versus Mdm2), the contribution of Mcl-1 and p53 in Mule-dependent DNA damage response, and regulatory mechanisms of Mcl-1 and p53 ubiquitination will be investigated in this aim. In Aim 3, we will further characterize the interaction of Mule with its substrates; especially map the binding site of p53 to Mule. We will study how the interaction between Mule and substrates are regulated by DNA damage. More importantly, we will search for novel protein factors modulating Mule acitivity through tandem affinity purification. In Aim 4, we will put more focus on post- translation modification of Mule, especially phosphorylation of Mule in DNA damage response and discuss potential mechanisms involved in it. These experiments should reveal novel mechanisms mediating DNA damage signals and how they are transduced through Mule, thereby providing fundamental insights into regulatory networks controlling apoptosis and DNA damage response. A mechanistic understanding of this pathway will help us decipher how apoptosis is deregulated in cancer and potentially identify new targets for therapeutic intervention.